The hematopoietic system produces the mature highly specialized blood cells known to be necessary for survival of all mammals. These mature cells include erythrocytes, specialized to transport oxygen and carbon dioxide, T- and B-lymphocytes, responsible for cell- and antibody-mediated immune responses, platelets or thrombocytes, specialized to form blood clots, and granulocytes and macrophages, specialized as scavengers and as accessory cells to combat infection. All of these specialized mature blood cells are derived from a single common primitive cell type referred to as the pluripotent stem cell found primarily in bone marrow.
The mature highly specialized blood cells must be produced in large numbers continuously throughout the life of a mammal. The vast majority of these specialized blood cells are destined to remain functionally active for only a few hours to weeks. Thus, continuous renewal of these mature blood cells, the primitive stem cells themselves, as well as any intermediate or lineage, committed progenitor cell lines lined between the primitive and mature cells, is necessary in order to maintain the normal steady state blood cell needs for continued life of the mammal.
At the heart of the hematopoietic system lies the pluripotent stem cell(s). These cells are relatively few in number and undergo self-renewal by proliferation to produce daughter stem cells, or they are transformed in a series of differentiation steps into increasingly mature lineage-restricted progenitor cells, ultimately forming the highly specialized mature blood cell(s).
The underlying principal of the normal hematopoietic cell system appears to be decreased capacity for self-renewal as multipotency is lost and lineage-restriction and maturity is acquired. Thus, at one end of the hematopoietic cell spectrum lies the pluripotent stem cell possessing the capacity for self-renewal and differentiation into all the various lineage-specific committed progenitor cells. At the other end of the spectrum lie the highly lineage-restricted progenitors and their progeny which have lost the ability of self renewal but have acquired mature functional activity.
The proliferation and development of stem cells and lineage-restricted progenitor cells are carefully controlled by a variety of hematopoietic growth factors or cytokines. Thus, hematopoietic growth factors may influence growth and differentiation of one or more lineages, may overlap with other growth factors in affecting a single progenitor cell-line, or may act synergistically with other factors.
It will be appreciated from the foregoing that novel hematopoietic growth factors that effect survival, proliferation, differentiation or maturation of any of the blood cells or predecessors thereof would be useful, especially to assist in the re-establishment of a diminished hematopoietic system caused by disease or after radiation- or chemo-therapy.
Platelets are critical elements of the blood clotting mechanism. Depletion of the circulating level of platelets, called thrombocytopenia, occurs and is manifested in various clinical conditions and disorders. Clinical thrombocytopenia is commonly defined as a condition wherein the platelet count is below about 150.times.10.sup.9 per liter. The major causes of thrombocytopenia can be broadly divided into three categories on the basis of platelet life span, namely: 1) impaired production of platelets by the bone marrow, e.g., thrombocytopenia brought about by chemo- and radiation-therapy, 2) platelet sequestration in the spleen (splenomegaly) and 3) increased destruction of platelets in the peripheral circulation, e.g., thrombocytopenia brought about by autoimmune disorders. Additionally, in patients receiving large volumes of rapidly administered platelet-poor blood products, thrombocytopenia may develop due to dilution factors. A more detailed description of thrombocytopenia and its causes, may be found in Schafner, "Thrombocytopenia and Disorders of Platelet Disfunction", Internal Medicine, John J. Hutton et al. Eds., Little, Brown & Co., Boston/Toronto/London, Third Ed. (1990) as well as International Patent Application No. PCT/US94/14553 (International Publication No. WO95/18858), referred to supra.
The therapeutic approach to the treatment of patients with thrombocytopenia is dictated by the severity and urgency of the clinical situation. The treatment is similar for HIV-associated and non-HIV-related thrombocytopenia, and although a number of different therapeutic approaches have been used, the therapy remains clinically controversial.
It will be appreciated from the foregoing that one way to treat thrombocytopenia would be to obtain an agent capable of accelerating the differentiation and maturation of megakaryocytes or precursors thereof into the platelet-producing form. Considerable efforts have been expended on identifying such an agent. One commonly referred to is thrombopoietin (TPO), the subject of the present application. Other names for TPO commonly found in the literature at this time include: thrombocytopoiesis stimulating factor (TSF); megakaryocyte colony-stimulating factor (MK-CSF), megakaryocyte growth and development factor, megakaryocyte stimulating factor, megakaryocyte potentiator and mpl ligand.
The cited International Patent Application PCT/US94/14553 describes the identification, isolation, production and use of an isolated mammalian megakaryocytopoietic proliferation and maturation promoting protein denominated the "MPL ligand" (ML), or more commonly, "thrombopoietin" (TPO), which has been found capable of stimulating proliferation, maturation and/or differentiation of megakaryocytes into the mature platelet-producing form.
Attention is directed as well to International Patent Application Publications Nos. WO95/26746, WO95/21919 and WO95/21920.
The PCT/US94/14553 application includes various aspects of associated embodiments of TPO, including a method of treating a mammal having or at risk for a hematopoietic disorder, notably thrombocytopenia, comprising administering a therapeutically effective amount of TPO materials to the mammal. Optionally, TPO is administered as such or in combination with a cytokine, especially a colony stimulating factor or interleukin. For purposes disclosed in said International Patent Application, TPO is broadly defined as including TPO itself or various variants, derivatives or isoforms thereof, including fragments that share at least one biological property in common with intact TPO for the treatment of thrombocytopenia. "Biological property", when used in conjunction with the definition of the various TPO materials useful as described in said patent application, means that they have thrombopoietic activity or an in vivo effector or antigenic function or activity that is directly or indirectly caused or performed by the TPO material.
With respect to the therapeutic use of thrombopoietin materials, as described in said International Patent Application No. PCT/US94/14553, the TPO materials are therein described for administration in admixture with a pharmaceutically acceptable carrier via any of several administrative modes. The daily regimen is described as ranging from about 0.1 to 100 .mu.g/kg body weight, preferably from about 0.1 to 50 .mu.g/kg body weight, preferably at an initial dosage ranging from about 1 to 5 .mu.g/kg per day. Implicit within the teachings of said patent application is a regimen of administering such a dosage rate over a period of several to many days following a projected or actual state of reduced platelet count.
Published clinical studies of clinically administered thrombopoietin indicates a dosage and administration regimen consisting of the administration of thrombopoietin, subcutaneously at dosages of 0.03 to 5.0 .mu.g/kg body weight once per day over a period of ten days for a condition marked by thrombocytopenia. See Abstract 1977, Blood 86 (1995). See also Abstracts 1012, 1014 and 1978, Blood 86 (1995).
Likewise, the compound epoetin alfa, which is a given name for erythropoietin (marketed as Epogen by Amgen, Inc.), is a glycoprotein indicated for stimulation of red blood cell production. It is indicated in a dosage and administration regimen consisting of starting doses over a range of 150 to 300 units per kg three times weekly for a period of many weeks in order to stimulate the proliferation of red blood cells in patients suffering from a depletion however realized.
Filgastrim, marketed as Neupogen by Amgen, Inc., is a granulocyte colony stimulating factor (G-CSF). Its indicated regimen is the administration of from 5 to 10 .mu.g/kg subcutaneously daily for two weeks.
Based upon this anecdotal evidence, the conventional regimen in administering materials for the proliferation of red blood cells or other primary blood cells to reverse the effects of thrombocytopenia, is continuous administration of therapeutically effective amounts of the biological material daily over a period of many days to patients in need of such therapy following an episode resulting in thrombocytopenia.
For convenience to physicians and especially patients alike, there exists an objective of developing alternative dosage/administration regimens of such biological materials that would be advantageous and therapeutically equivalent or superior to reverse the effects of thrombocytopenia.